Griselimycin is a naturally occurring antibiotic produced by microorganism of the genus Streptomyces which is the largest genus of the Actinobacteria and the type genus of the family Streptomycetaceae. Streptomycetes are Gram-positive and have genomes with high GC-content. Streptomycetes are characterized by a complex secondary metabolism. They produce over two-thirds of the clinically useful antibiotics of natural origin. Griselimycin is an antibiotic which was first described by Terlain and Thomas, 1971. It is composed of ten amino acids being in this order L-N-methylvaline, which is N-acetylated, L-trans-4-methylproline, L-N-methylthreonine, L-leucine, L-trans-4-methylproline, L-leucine, L-N-methylvaline, L-proline, D-N-methylleucine and glycine. The peptide chain is cyclised between glycine and L-N-methylthreonine via an ester bond resulting in a non-cyclised tail consisting of the amino acids L-N-methylvaline, which is N-acetylated, and L-trans-4-methylproline.
Griselimycin belongs to the group of non-ribosomally synthesized microbial peptides which group of peptides shows a remarkable structural diversity and comprises a wide-spread class of the most potent antibiotics and other important pharmaceuticals. Although structurally diverse, non-ribosomally synthesized microbial peptides share a common mode of synthesis, the multienzyme thiotemplate mechanism. Thereby, peptide bond formation takes place on large multienzyme complexes, which simultaneously represent template and biosynthesis machinery. These multienzyme complexes are designated non-ribosomal peptide synthetases (NRPSs). Sequencing of genes encoding NRPS revealed a modular organization. A module is a section of the NRPS polypeptide chain that is responsible for the incorporation of one defined monomer into the growing polypeptide chain. Thus, NRPSs are used simultaneously as template because the amino acid to be incorporated is determined by the module and as biosynthetic machinery because it is the module that harbors all necessary catalytic functions. Modules can be further dissected into catalytic domains. These domains catalyse at least the steps of substrate activation, covalent binding and peptide bond formation. Domains of equal function share a number of highly conserved sequence motifs. They can be modified for activity changes within the polypeptide chain which opens up prospects for manipulation of the NRPS machinery.
Within each module, the selection of a specific substrate (amino acid) is mediated by an adenylation domain (A-domain). This A-domain is responsible for the selection of the amino acids that make up the product and thus controls its primary sequence. A-domains activate amino and also some carboxy substrates as amino acyl adenylate while ATP is consumed. This reactive intermediate is further transported onto the terminal cysteamine thiol moiety of the Ppan prosthetic group that is attached to the peptidyl carrier protein (PCP) domain which is also referred to as thiolation (T) domain located downstream of the A-domain in the same module. It represents the transport unit that accepts the activated amino acid that is covalently tethered to its 4′PP cofactor thioester. This cofactor acts as a flexible arm to allow the bound amino acyl and peptidyl substrate to travel between different catalytic centers. The combination of A-domain and T-domain is defined as an initiation module since both domains are required to activate and covalently tether the first building block for subsequent peptide synthesis. The initiation modules of lipopeptide pathways harbor an additional N-terminal C domain for condensation of an acyl side chain with the amino group of the first amino acid. The condensation or C-domain is the central entity of non-ribosomal peptides synthesis because it is responsible for peptide bond formation between amino acyl substrates bound to the T-domain of adjacent modules. The enzyme catalyses the nucleophilic attack of the amino (or imino, hydroxyl) group of the activated amino acid bound to the downstream (with respect to the C-domain) module onto the acyl group or the amino acid tethered to the upstream module. The resulting peptidyl intermediate is then translocated down the assembly line for subsequent condensation and further modification steps. During synthesis, the growing peptide chain is handed over from one module to the next module until it reaches the final modul's T-domain. This module contains in most cases a TE (thioesterase)-domain that is important for the liberation of the product. Product release is achieved by a two-step process that involves an acyl-O-TE-enzyme intermediate that is subsequently attacked by either a peptide-internal nucleophile or water, which results in a macrocyclic product or a linear peptide, respectively.
An especially striking feature of non-ribosomal peptides is the occurrence of D-amino acids. One way to incorporate a D-amino acid is to use a D-amino acid selective A domain. The more common way is, however, through the use of E (epimerization)-domains that occur at the C-terminal end of modules responsible for the incorporation of D-amino acids. The enzyme catalyses the epimerisation of T-domain bound L-amino acid of the growing peptide chain. Discrimination of the T-domain bound L-amino acid through enantioselectivity of the downstream condensation domain donor site leads to a clean D-configurated product.
A number of NRPS contain methyltransferases (MT domain) that are responsible for the N- or C-methylation of amino acid residues thus making the peptide less susceptible to proteolytic breakdown. Both types of methyltransferases (N- or C-methyltransferases or abbreviated N-MT or C-MT) use S-adenosyl methionine as the methyl donor. The MT domains are often found as insertions in the A-domains (Finking et Marahiel, 2004; Marahiel, 2009). Even more striking is the incorporation of unnatural amino acids such as methyl-proline which are usually biosynthesized in advance to their use in the assembly line. Alternatively, incorporated amino acids can be modified enzymatically post assembly by the NRPS.
Griselimycin is naturally produced by microorganisms of the genus Streptomyces. However, although the production of griselimycin by Streptomyces has been known in the art, the genetic locus responsible for the biosynthesis of griselimycin has not been identified so far. Consequently, targeting and modification of the biosynthesis of griselimycin were still limited. Accordingly, there is a need for genetic information regarding the biosynthesis of griselimycin.